CD4⁺CD3⁻ cells are the predominant hematopoietic cells found in mouse fetal intestine. We prove their role as Peyer's patch (PP)-inducing cells by transfer into neonatal PP-deficient mice. To test the requirement of chemokines and adhesion molecules in induction of PP, we studied mice deficient in CXCR5 and/or α4β1 integrin-mediated adhesion. CXCR5^−/− mice have CD4⁺CD3⁻ cells, which are inefficient in inducing PP formation. We show here that CXCR5/CXCL13 signaling activates α4β1 integrin on CD4⁺CD3⁻ cells. Blocking of β1 integrin or VCAM-1, the ligand of α4β1 integrin, inhibits PP formation. This study demonstrates the link between chemokine receptors and adhesion molecules that regulates stromal/hematopoietic cell interaction leading to PP formation.